Jingming Zhou,Xiaoli Zhang,Wenjing Qian,Qingbao Yang,Yanhua Qi,Yumei Chen,Aiping Wang
Tiamulin (TML) is a kind of diterpenoid veterinary antibiotic. If abused, it will cause residues in edible tissues of animals and endanger human health. Thus, a facile, sensitive, and accurate method is essential for TML detection. In this study, TML complete antigen was synthesized by oximation and active ester method, and monoclonal antibodies with high specificity and sensitivity were prepared. ZnCdSe/ZnS quantum dots (QDs) coupled with goat anti-mouse IgG were used as a fluorescence detection probe to establish indirect competitive fluorescent immunosorbent assay (icFLISA) for the determination of TML in pork. The 50% inhibitory concentration (IC 50 ) of the icFLISA was 0.069 ng/ml, and the limits of detection (LOD) were 0.309 pg/ml. The recovery of the spiked pork samples for the icFLISA ranged from 85.6 to 96.8%, and the coefficient of variation (CV) was less than 9.4%. The results showed that the icFLISA based on ZnCdSe/ZnS QDs was more sensitive than the traditional indirect competitive enzyme-linked immunosorbent assay for the detection of TML in edible animal tissues.
Novel pleuromutilin derivatives designed based on the structure of valnemulin were synthesized and evaluated for their in vitro antibacterial activities. These pleuromutilin derivatives with substituted amino moiety exhibited excellent activities against methicillin-resistant Staphylococcus aureus, methicillin-resistant Staphylococcus epidermidis, Escherichia coli, and Streptococcus agalactiae. Compound 5b showed the highest antibacterial activities and even exceeded tiamulin. Moreover, the docking experiments provided information about the binding model between the synthesized compounds and peptidyl transferase center (PTC) of 23S rRNA.
The binding of tiamulin with calf thymus DNA was systematically investigated using multispectroscopy and molecular modelling techniques. For DNA, once tiamulin was added, viscosity ( η ) and melting temperature ( T m ) both exhibited an uptrend. The fluorescence performance of the tiamulin–DNA complex did not change with the ionic strength changes. The binding constant ( K a ) of tiamulin for single-stranded DNA (ssDNA, 1.48 × 10 4 M −1 ) was obviously higher than that for double-stranded DNA (dsDNA, 9.51 × 10 3 M −1 ) at 291 K. The helix structure became looser and the base stack force became stronger for DNA due to the presence of tiamulin as seen from circular dichroic (CD) spectra. The intercalation binding mode of tiamulin with DNA was disclosed. Molecular modelling also revealed tiamulin inserting into the base pairs with the lowest binding free energy of −18.73 kJ mol −1 using van der Waals forces as well as hydrogen bonds.
Valnemulin (VAL) and tiamulin (TIA) are pleuromutilin antibiotics used primarily for treating bacterial infections in swine or other food animals. Furthermore, VAL and TIA are also employed as feed additives to promote animal growth. However, the illegal use of VAL and TIA could cause a series of hazards to consumers. Here, VAL was designed to be conjugated with bovine serum protein to prepare immunogen. A highly sensitive monoclonal antibody that recognized both VAL and TIA has been successfully produced. Moreover, an immunochromatographic strip assay for rapidly screening VAL and TIA in porcine liver was established with visual detection limits (cutoff values) of 50 and 25 ng/g, respectively. The IC 50 values calculated from the equation of the standard curve were 6.06 and 3.45 ng/g and the limits of detection were 0.96 and 0.29 ng/g for VAL and TIA. According to the recovery experiment results, the test strip exhibited acceptable accuracy and precision. Generally, the proposed strip provided a practical tool for the detection of VAL and TIA. Practical Application We produced a highly sensitive monoclonal antibody and developed an immunoassay strip for simultaneously monitoring TIA and VAL. Additionally it was preliminarily confirmed that the rapid detection tool was suitable for screening TIA and VAL in porcine liver.
危险品化学品经营许可证（带存储）